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The effect of muscimol, a GABAA receptor agonist, on the basic metabolic activity was investigated in the mouse brain and was correlated with its receptor occupancy. For the quantitative evaluation of the functional activity of the mouse brain, the cerebral glucose utilization was measured by the double tracer technique, using [14C] 2-deoxyglucose and [3H]3-O-methylglucose. The dose-dependent reduction in the cerebral glucose utilization was observed after intravenous administration of various doses of muscimol (0.3-1.5 mg/kg). On the other hand, the GABAA receptor occupancy of muscimol was determined by using the values of the unbound drug concentration in the brain tissue and the receptor dissociation constant based on the in vitro binding experiments using the dissociated brain cells. The tissue unbound concentration of muscimol was calculated by multiplying the total concentration in the brain after administration of muscimol and the tissue unbound fraction, which was measured by the equilibrium dialysis method using brain tissue homogenate. A linear relationship was observed between the GABAA receptor occupancy of muscimol and the decrease in the cerebral glucose utilization. This finding indicates that the simple receptor occupancy theory holds for this receptor-ligand system, and there is a large difference in the effect on glucose metabolic response between GABAA receptor-agonist interaction and benzodiazepine receptor-agonist interaction.

Kinetic analysis of valproate (VPA) disposition in pregnant rats was performed. A dose-dependent saturable plasma elimination was observed in both the control and pregnant rats. Saturation was more remarkable in the pregnant rats and this was assumed to be due to the decreased hepatic extraction by metabolism. Pharmacokinetic parameters were calculated using a two-compartment open model with a Michaelis-Menten-type elimination process from the central compartment. In the pregnant rats, the calculated values of Km and Vmax decreased significantly, by one-half of those of the control rats, whereas no significant difference was observed in distribution rate constants (k12, k21) and volumes of distribution (V1, Vdss) between two groups of rats. Furthermore, the hepatic extraction of VPA in the control and pregnant rats was investigated in order to explain nonlinear pharmacokinetics of VPA. The values of the hepatic extraction ratio in the control and pregnant rats were 39.9 and 22.4% at the steady state concentration of approximately 20 micrograms/ml, respectively. The extraction ratios in the pregnant rats were lower than those of the control rats. This fact may be one of the reasons why the elimination of VPA in the pregnant rats is more susceptible to saturation.

The effects of salicylic acid on the pharmacokinetics of valproic acid were investigated in bile-exteriorized rats. A 50 mg/kg bolus dose of sodium valproate was injected iv to Long Evans rats with and without (control) prior treatment by constant infusion of salicylate to keep it at steady state plasma level (about 250 micrograms/ml). The plasma elimination of valproic acid followed a monoexponential decline in both salicylate-treated and control rats. A significant increase (p less than 0.01) in the disposition rate constant (kel), the volume of distribution (Vd), and the total clearance (Cltot) as well as a significant decrease (p less than 0.01) in the AUC and the elimination half-life (t1/2) were observed in the salicylate-treated rats. In spite of the significantly lowered total plasma level and increased unbound fraction of valproic acid in the salicylate-treated rats, there were no significant differences in unbound valproic plasma levels and unbound valproate pharmacokinetic parameters. The biliary excretion of unchanged and conjugated valproate was not significantly different between the two groups. The in vitro plasma-unbound fractions (fu) of valproic acid were significantly increased (p less than 0.01) in the presence of salicylic acid. The apparent dissociation constant of plasma protein binding for valproic acid was increased from 0.287 to 1.204 mM in the presence of salicylic acid. These findings indicate that the pharmacokinetic changes of valproic acid in the presence of salicylic acid were consistent with the elevation in the plasma-unbound fraction of valproic acid due to displacement from plasma protein-binding sites by salicylic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to simulate the distribution and elimination of radioiodinated human beta-endorphin (125I-beta-EP) after iv bolus injection in rats, we proposed a physiologically based pharmacokinetic model incorporating diffusional transport of 125I-beta-EP across the capillary membrane. This model assumes that the distribution of 125I-beta-EP is restricted only within the blood and the tissue interstitial fluid, and that a diffusional barrier across the capillary membrane exists in each tissue except the liver. The tissue-to-blood partition coefficients were estimated from the ratios of the concentration in tissues to that in arterial plasma at the terminal (pseudoequilibrium) phase. The total body plasma clearance (9.0 ml/min/kg) was appropriately assigned to the liver and kidney. The transcapillary diffusion clearances of 125I-beta-EP were also estimated and shown to correlate linearly with that of inulin in several tissues. Numerically solving the mass-balance differential equations as to plasma and each tissue simultaneously, simulated concentration curves of 125I-beta-EP corresponded well with the observed data. It was suggested by the simulation that the initial rapid disappearance of 125I-beta-EP from plasma after iv injection could be attributed in part to the transcapillary diffusion of the peptide.

Nonlinear and slow tissue distribution of ouabain was found in rabbits, and the contribution of the ouabain binding to Na+,K+-ATPase was also examined in the heart. The tissue-to-plasma concentration ratio (Kp) of ouabain in each tissue increased with time until 120 min. In addition, the Kp also depended on the plasma concentration (Cp). In the heart, Kp decreased from 12.6 +/- 1.2 to 1.9 +/- 0.2 with the increase of the Cp from 67.3 +/- 5.0 to 497 +/- d 85.2 nM. These data indicate that ouabain distributes slowly into each tissue by a saturable process. The specific binding of ouabain to cardiac Na+,K+-ATPase was measured using crude homogenates. The dissociation constant and the binding capacity were 86 nM and 2.3 nmol/ml of 0.5% homogenate, respectively. The tissue-to-plasma concentration ratio (Kp,vitro) was constructed based on these binding parameters and this explained almost 40% of the Kp obtained in vivo. These results suggest that the ouabain binding to Na+,K+-ATPase might play one of the major roles in the slow and nonlinear tissue distribution of ouabain in rabbits.

Time courses of anticonvulsive effect nd benzodiazepine receptor occupation in the brain were determined after iv administration of diazepam (DZP) (1.2 mg/kg) to rats. Score for the anticonvulsive effect was assigned to each DZP-treated rat depending on the degree of protection against pentylenetetrazole-induced seizures. Measurement of in vivo receptor occupation was made by comparing the specific 3H-DZP binding in vitro to the crude synaptosomal fractions between vehicle-treated rats (control) and DZP-injected rats. The receptor occupation declined in parallel with the anticonvulsive effect with approximate half-life of 1 hr. A good linear correlation (r = 0.977) was observed between the receptor occupation and the anticonvulsive effect. The time course of anticonvulsive effect was further compared with that of DZP concentration in the brain which was previously reported [Igari et al., Drug Metab. Dispos. 10, 676 (1982)]. The anticonvulsive effect was nonlinearly related to the DZP concentration in the brain with the half-maximum concentration, Kd in vivo of 793 nM. The half-maximum concentration for the unbound DZP, Kd in vivo was also estimated to be 113 nM, which was still 50 times the values of Kd (2.3 nM) determined at 0 degree C from an in vitro binding study of 3H-DZP to the receptor in the crude synaptosomal fraction.

The interaction between tolbutamide (TB) and sulfonamide (SA) in rabbits was quantitatively investigated by both in vivo and in vitro experiments, and the mechanisms of species difference between rabbits and rats were analyzed by comparing the two total body clearances (CLtot) obtained from in vivo and in vitro studies. The sulfonamides used were sulfaphenazole (SP) and sulfadimethoxine (SDM). In vivo CLtot of TB was changed little by SA in rabbits, which was contrary to the phenomenon seen in rats, i.e. CLtot was markedly decreased by SA in rats (Sugita et al., Biochem. Pharmacol., 30, 3347, 1981). The total body clearance defined as CL in vitro tot.pred was predicted for TB in the presence and absence of SA by the equation: CL in vitro tot.pred congruent to fB CL in vitro int, where fB is the blood-free fraction and CL in vitro int is the hepatic intrinsic clearance of unbound drug obtained from in vitro experiments using liver microsomal fraction. A comparatively good agreement was observed between the two CLtot obtained from in vivo and in vitro data. The analysis in rabbits based on these in vitro experiments showed that the small gross changes of in vivo CLtot induced by SA were due to the compensative changes of two factors, i.e. fB and CL in vitro int which in the presence of SA were approximately 200 and 50% of the control, respectively. However, in rats, the marked decrease of in vivo CLtot induced by SA was explained by the greater degree of the decrease in CL in vitro int than that of the increase in fB.(ABSTRACT TRUNCATED AT 250 WORDS)

Our previous studies using an in vivo tissue-sampling single-carotid injection method have shown that the transport of DL-propranolol into rat brain is inhibited by the serum from rats with uranyl nitrate-induced acute renal failure. The present studies were designed to examine the effect of serum from patients with renal or liver disease on the transport of DL-propranolol into the rat brain. While the binding of DL-propranolol to serum from cirrhotic patients was significantly decreased compared to normal serum, there was no change for the serum from patients with renal failure. In the carotid injection studies, the brain transport parameters such as the brain uptake index (BUI), the unidirectional extraction ratio (ET), the blood-brain barrier permeability surface area product (PSapp), and PSapp corrected for the unbound fraction (PSuapp) in rats injected with serum from patients with renal failure were significantly reduced to approximately 40-53% of those in controls. No change in BUI, ET, and PSapp was found in rats injected with serum from cirrhotic patients. However, the cirrhotic patients adopted in the present study had relatively mild liver disease (judging from the biochemical blood test), and we cannot refer to the more severe cirrhotic patients only from this study. Moreover, significant correlations were observed between the biochemical parameters (blood urea nitrogen, serum creatinine concentration) representing the degree of renal failure and the transport parameters (ET, PSapp, or PSuapp) of DL-propranolol.(ABSTRACT TRUNCATED AT 250 WORDS)

Recent kinetic studies using in situ perfused rat liver suggested that the hepatic uptake of extensively albumin-bound ligands is mediated primarily by direct interaction of the albumin-ligand complex with the hepatocyte surface rather than by the small unbound fraction of ligand, as has been generally believed [Ockner et al., Am. J. Physiol. 245, G13 (1983)]. In order to investigate this mechanism in vivo, rose bengal (RB) was injected iv to the normal and Nagase analbuminemic mutant rats (NAR) and both the pharmacokinetic parameters and the serum protein binding parameters for the two groups were compared. The serum disappearance curves of RB in normal rats and NAR were almost superimposed, and no significant difference in various pharmacokinetic parameters including the hepatic uptake clearance (k12V1) was observed between the two groups. Nevertheless, the unbound fractions of RB in serum were approximately 4-fold (equilibrium dialysis method) and 10-fold (spectrophotometric method) higher than those in normal rats. However, in both groups of rats RB is extensively bound to plasma proteins and more than 99.8% of RB in the plasma exists as the protein-bound form. The intrinsic ability of the two groups of rats to take up unbound RB was compared using isolated liver cells. No significant difference between the two groups was observed in the initial velocity of uptake. From these findings, we concluded that the hepatic uptake of RB is primarily driven by the serum protein-bound form and not by the unbound form and that the serum protein-mediated uptake mechanism of RB was not specific only for serum albumin but also for other serum proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

We have kinetically investigated the tissue distribution of 14C-diazepam and described the appearance and disappearance of its metabolites (3-hydroxydiazepam, desmethyldiazepam, and oxazepam) following a single iv injection of 14C-diazepam into rats. Significant amounts of oxazepam were detected in plasma and various tissues in the rat, contrary to previous reports. Concentration-time profiles of diazepam in the main disposing organs (liver, kidney, and lung) and the other organs (brain, heart, and small intestine) indicated that diazepam was distributed rapidly to these organs. Concentration-time profiles of diazepam in the main tissues for drug distribution (skin and adipose) indicated that diazepam was slowly distributed to these tissues, whereas that in muscle, which is also responsible for drug distribution, indicated that diazepam was less rapidly distributed to this tissue. Metabolites appeared in plasma and various tissues or organs immediately after iv injection of diazepam. Metabolites levels in plasma and various tissues or organs were significantly lower than that of diazepam except for liver and small intestine, where metabolites levels were higher compared to that of diazepam and metabolites exhibited a considerable persistence.

In our previous study [Lin, Sugiyama, Awazu, and Hanano: J. Pharmacokin. Biopharm. 10,649 (1982)], we successfully applied a physiological pharmacokinetic model to quantitative prediction of the elimination and distribution kinetics of ethoxybenzamide in rats and rabbits. The predictions of the time course of ethoxybenzamide concentrations in plasma were good at lower doses (10 and 20 mg/kg), whereas those at high dose (80 mg/kg) were poor. In the present study, therefore, product inhibition was suspected and examined. Product inhibition of ethoxybenzamide deethylation by its metabolite, salicylamide, was demonstrated both in vivo and in vitro studies. The plasma disappearances of ethoxybenzamide after a 20 mg/kg iv injection were determined both in the control and the salicylamide-treated rabbits. In the salicylamide-treated rabbits, the plasma disappearance of ethoxybenzamide was significantly delayed compared to that in control rabbits. This delay was quantitatively explained by the physiological pharmacokinetic model taking the competitive-type of product inhibition into consideration. The apparent dissociation constant for the salicylamide-enzyme complex in vivo was estimated as 0.14 mM. The inhibition of ethoxybenzamide de-ethylation by salicylamide was observed also in in vitro study using liver microsome of rabbits.

The effect of pregnancy on tissue distribution of salicylate was studied by comparing both pharmacokinetic and protein-binding parameters between 20-day-pregnant rats and nonpregnant (control) rats. In the pregnant rats, the volume of distribution increased significantly (p less than 0.05) from 164 ml/kg of the control to 225 ml/kg, and the total body clearance also increased significantly (p less than 0.05) from 12.1 ml/hr/kg of the control to 19.8 ml/hr/kg. But these changes did not affect the plasma disappearance half-life of salicylate in the pregnant rats. The serum unbound fraction (fs) of the pregnant rats at 8 hr after iv administration of salicylate increased remarkably from 0.14 of the control to 0.67. The fs in the fetal serum (0.41) was lower than that in the maternal serum in spite of the lower albumin concentration in the fetal serum. A nonlinear serum protein binding was observed both in the control and in the fetal rats, but not observed in the pregnant rats. In the pregnant rats, the tissue-to-serum concentration ratios (Kp) of all tissues studied were larger than those in the control rats, and the values of Kp in the fetal were larger than those in the maternal. To elucidate these difference in Kp values between the pregnant and control rats, a mathematical model was proposed, where salicylate was distributed in the interstitial fluid, bound to the interstitial albumin, and translocated into the intracellular fluid according to the pH partition theory. The Kp values of most tissues in the control and pregnant rats were predicted successfully by using this model.(ABSTRACT TRUNCATED AT 250 WORDS)